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Heterofermentative lactic acid bacterium Leuconostoc mesenteroides ferments sucrose from the semi-defined medium [Vandamme J.E., Van Loo J., Simkens E., De Laporte A. Optimization of sucrose phosphorylase production by Leuconostoc mesenteroides. J. Chem Tech. Biotechnol. 1987, 39, 251-262] and produces mainly lactic acid and acetic acid, as followed by a HPLC method. Relatively fast production of these acids reduces a pH value of broth for at least two pH units to pH3.0 and makes it almost completely unsuitable for growth of potential contaminants. Growth pattern of L. mesenteroides is strongly affected by the availability of oxygen and at the laboratory- scale concentration of bacterial biomass is about 40% higher under aerobic conditions than under microaerophilic conditions. According to available data, this Gram-positive bacterium possesses a gene encoding sucrose phosphorylase (EC 2.4.1.7) that catalyzes transglucosylation reaction in which sucrose can be used as a glucosyl donor and glycerol as a glucosyl acceptor, and the reaction results in a production of glucosylglycerol and fructose. Possible production of glucosylglycerol by permeabilized cells of L. mesenteroides was investigated in the laboratory-scale. L. mesenteroides cells pregrown in the semi- defined medium at 29±1C, 200 rpm and nonmaintained pH value were harvested when they were in the middle of exponential growth phase and transferred to reaction mixture containing 0.8 M sucrose and 2.0 M glycerol. pH value of resulting suspension was adjusted to 5.0 (1.0 M NaOH) and then the cells were permeabilized by continuous ultrasound treatment (37 kHz, 90 W, 29±1℃ over 4 hours) or periodic ultrasound treatment (37 kHz, 90 W, 29±1℃ two times over 30 minutes with 30 minutes pause inbetween the each treatment). Besides, surfactant Tween-80 (0.4% and 0.6%) was used as cells permeabilization agent. After permeabilization of the cells they retained their fermentative activity, grew in linear mode under above decribed conditions and again reduced pH value of the mixture to pH3.0. In HPLC chromatograms of withdrawn supernatants unknown compound was eluted between sucrose and fructose and this retention time do not correspond to any identified compound of the mixture that was used in glucosylation experiments. In order to identify the unknown compound, its separation and further identification is required. |
